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I attended your ChIP webinar last February.I am currently optimizing a ChIP-seq protocol with my cells (murine primary cells), with an antibody against Pol-II (product ab817). Before going on with the sequencing part, I would like to make sure that my immunoprecipitation worked well. As you suggested in your Webinar, I am looking for positive and negative control loci that I will test first by qPCR. Would you have any suggestions for negative control regions for Pol-II binding ? Have you ever used telomeric or intergenic regions for example, or would you rather recommend non-expressed genes in the tissue under study ? I also saw in some cases people using negative ascites as control. What is that for ?
Thank you very much in advance for your help.
Asked on Apr 19 2012
Thank you for contacting us.
We have tested some of our RNA polymerase II antibodies against both inactive genes and the 3' end of active genes. Below is a link to the antibodies.
We also stock a number of pre designed primers to human regions in our catalogue.
Reviewing the antibody datasheets there are a number of regions and primer sets that might be suitable.
I have not seen people using negative ascites as a control but I assume this is a negative control for the IP part of ChIP. It is also possible to use no antibodies or a non-specific antibody that is from the same host species and isotype. e.g. use a rabbit IgG if this matches your primary antibody.
I hope this helps. Please let me know if there is any other information you need.
Answered on Apr 19 2012